LncRNA PANTR1 is Associated with Poor Prognostic and Suppresses Apoptosis in Glioma

Glioma is the most common tumor in the central nervous system. High-grade gliomas confer a poor prognosis, being a serious health and economic burden. Current literature suggests the important role of long noncoding RNA (lncRNA) in mammals, especially in tumorigenesis of various tumors. The functions of lncRNA POU3F3 adjacent noncoding transcript 1 (PANTR1) have been investigated in hepatocellular carcinoma but remain yet unclear in gliomas. We evaluated the role of PANTR1 in glioma cells using published data from The Cancer Genome Atlas (TCGA), then validated it by ex vivo experiments. To investigate the potential cellular mechanism of different levels of PANTR1 expression in glioma cells, we used siRNA-mediated knockdown in low-grade (grade II) cell lines and GBM (grade IV) cell lines (SW1088 and SHG44, respectively). On the molecular level, low expression of PANTR1 caused significantly reduced glioma cell viability and enhanced cell death. Moreover, we identified the importance of PANTR1 expression for cell migration in both cell lines, a critical foundation for invasiveness in recurrent gliomas. In conclusion, this study provides the first evidence that PANTR1 has a relevant role in human glioma by influencing cell viability and cell death.


Introduction
Glioma is the most common primary brain tumor, accounting for 81% of malignant intracranial tumors and resulting in severe morbidity and mortality [1]. Glioblastoma (GBM), the most prevalent glioma histology (∼45% of all gliomas), remains a poor median survival time which is 12-15 months, despite advancements in treatment techniques such as surgery, chemotherapy, and radiotherapy [2][3][4], since of substantial drug resistance and a near-total recurrence rate [5]. To achieve a favorable prognosis, it is essential to establish novel therapeutic interventions. Plenty of research has suggested that extracellular vehicles (EVs) may hold great potential for creating more efective therapies for tumor patients [6,7]. Tese fndings revealed an increasing recognition of long noncoding RNAs (LncRNAs) as crucial mediators of EV biological efects [6].
lncRNAs are defned as any RNA with a length of more than 200 bps but with no obvious coding function [8]. Tey have diverse roles in tumorigenesis, cell viability, cell death, as well as cell migration and invasiveness [9,10]. Several recent studies have indicated that lncRNAs are related to the response of immunotherapy, TMZ resistance, and radiotherapy resistance, which could further afect the therapeutic outcomes of glioma patients [11,12]. Nowadays, applications of microarray technology and bioinformatic analysis have enabled a comprehensive investigation for identifying novel functional RNAs [13]. Especially the regulatory role of lncRNAs in tumor cell microenvironment and tumor growth has received a lot of attention. Although previously published studies suggested that some lncRNAs may have been associated with gliomas, the specifc characteristics by which lncRNAs act on glioma are not yet fully understood [14,15]. Te mechanism underlying the interaction between lncRNAs and glioma remains unknown.
Bian et al. found that glioma cells-derived sEVs delivering lncRNA-ATB can activate astrocytes by inhibiting miR-204-3p, and the activated astrocytes, in turn, promoted glioma cell invasion [16]. LncRNA AHIF, the natural antisense transcript of hypoxia-inducible factor 1α (HIF 1α), was reported to promote glioma cell proliferation, invasion, and radioresistance via sEVs and may act as a potential therapeutic target [17,18]. Chai and colleagues found that lncRNA ROR1-AS1 was up-regulated in glioma tissues in comparison with normal tissue, and high expression of lncRNA ROR1-AS1 presented a poor prognosis. Further research showed that sEVs derived from tumor cells and decorated with lncRNA ROR1-AS1 could facilitate glioma progression via inhibiting miR-4686 [19].
POU3F3 adjacent noncoding transcript 1 (PANTR1) is an oncogenic long noncoding RNA that is located at 4 kb upstream of the protein-encoding POU3F3 gene that has a substantial infuence on a variety of cellular features in various types of cancer. PANTR1 was initially found to be associating with the regulator of cortical development [20]. However, its role in tumorigenesis has not been fully investigated. Tus, the present study aimed to explore a novel interactome, PANTR1, and to discover the inner relationship between PANTR1 and gliomas. Considering PANTR1 has previously been reported to be associating with tumorigenesis of hepatocellular carcinoma, we hypothesize that PANTR1 may play a signifcant role in glioma tumorigenesis [21].

Material and Methods
2.1. Data Acquisition from TCGA. We started with data downloaded from GBM and LGG collection of TCGA datasets (https://portal.gdc.cancer.gov/). RNA-Seq data and their corresponding clinical data were obtained in Level 3 HTseq-FPKM format, and RNAseq data without corresponding clinical data were discarded. Tere were 670 cases with RNAseq data after the exclusion of cases without clinical data. For sample size calculation, we used Open Source Epidemiologic Statistics for Public Health (OpenEpi) to show that a size of at least 663 cases could achieve a 99% confdence level. RNAseq data in FPKM (fragments per kilobase per million) format were converted into TPM (transcripts per million reads) format for expression comparison among samples. Te data of WHO grade, IDH mutation, and 1p/19q codeletion status were obtained from the study of Ceccarelli et al. [22].
Te expression levels of PANTR1 in 5 normal adjacents to tumor (NAT) samples and 670 glioma samples of GBM and LGG collection were compared. Similarly, the expression levels of PANTR1 in tumor tissues were compared with NAT tissues of GTEx combined with TCGA.

Collection of Glioma
Tissues. For PANTR1 expression comparison, glioma tissues (n � 15) and normal adjacent tissues (n � 15) were collected from Shanghai General Hospital between February 2017 and October 2021, consisting of 12 grade II/III glioma patients and 3 GBM patients. Inclusion criteria included: (i) patients who underwent gross total resection and postoperative pathology diagnosed as glioma; (ii) complete patient information acquired. Exclusion criteria included: (i) patients who underwent any preoperative therapies such as radiotherapy and chemotherapy; (ii) Patients with multiple histological malignant tumor history; (iii) Patients with severe heart, lung, liver, spleen, or kidney diseases. Tis study was performed under the approval of local medical ethics (No. 2020SQ119) Shanghai General Hospital. Te processing of clinical tissue samples complies with the ethical standards of the Declaration of Helsinki. Informed consent of tissue sample donations for biomedical research was signed by all patients.

RNA Extraction and Reverse Transcription-Quantitative
(RT-q) PCR. RNAs were extracted from the glioma tissue, the NAT tissues, and SHG44 and SW1088 cell lines using a standard TRIzol protocol following the manufacturer's instructions. A High-Capacity cDNA Reverse Transcription Kit (Aidlab, Beijing, China) was used for cDNA synthesis following the manufacturer's protocol. A 7500 Real-Time PCR System (Applied Biosystems, CA, USA) was used for qRT-PCR with an SYBR Green PCR Kit (Roche, USA). Te expression fold-change was determined using the 2 −ΔΔCT .

Wound-Healing Assay.
Te migratory ability of glioma cells was evaluated by wound-healing assay. Cells were cultured in 6-well plates (1 × 10⁵/well) until 100% confuence was achieved. Ten, the cells were wounded using a plastic pipette tip, after which the cells were washed with PBS and incubated for 24 h. Tereafter, the wounded areas were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Cell Migration and Invasion
Assays. Te cell migration and invasion assays were carried out with 6-well Transwell insert chambers (Corning, NY, USA). Cells (5 × 10 4 ) were cultured in the upper Matrigel chamber. After 48 h of incubation at 37°C, the cells from the upper chamber were removed by cotton swabs, and the residual cells in the bottom chamber were fxed with methanol and stained with 1% crystal violet for 30 min. Cells were then enumerated using a microscope.

Annexin V/Propidium Iodide Assay.
To perform fow cytometric analysis, death cells were harvested using an Annexin V Cell death Detection Kit (BD Biosciences, CA, USA). Cells in the log phase were plated in 6-well plates at a density of 2 × 10 5 cells/well. In diferent groups, glioma cells were harvested using the Accutase ™ cell detachment solution (Sigma-Aldrich, USA) and then labeled with V-FITC reagents following the protocol of BD Bioscience. Te stained cells were analyzed via fow cytometry and enumerated by FACSDiva software.

Western Blot Analysis.
For Western blot analysis, we transferred total protein to a polyvinyldifuoridene membrane via RIPA lysis bufer. Protein expression was tested using the BCA protein assay. Total protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% resolving gel) and then electrotransferred to nitrocellulose membranes. Te primary antibodies for total or p-akt (ser 473) (Cat No.: # 5012), AKT (Cat No.: # 9272), and β-actin (Cat No.: # 3700s) rabbit mAb were obtained from Cell signaling Technology (CST), which have already been validated by the CST company. Rabbit anti-goat IgG horse-radish peroxidase-conjugated secondary antibodies were used for these experiments. We controlled the loaded amount of each lane and validated the expression of β-actin in the WB study. Te immunoreactive bands were captured on an X-ray flm as described previously [1]. Te western blot assay was performed 6 times.

Statistical Analysis.
All the statistical analysis processes were verifed by independent authors who were blinded to the experimental groups at least three times. Te tumor cells were grouped randomly. To compare two groups of continuous variables, the statistical signifcance of normally distributed variables was estimated using the independent Student's ttest, and the diferences between non-normally distributed variables were analyzed using the Mann-Whitney U test (i.e., Wilcoxon rank sum test). Chi-square or Fisher's exact tests were used to compare and analyze the statistical diferences between the two groups of categorical variables. Te sample size was determined by Shapiro-Wilk test. Pearson correlation analysis calculated the correlation coefcients between diferent gene sets. Te survival package in R was used for survival analysis. Te Kaplan-Meier survival curve was used to show the survival diference. Te log-rank test was employed to determine the signifcance of diferent survival times between the two groups. Univariate and multivariate Cox analyses were used to determine independent prognostic factors. Statistical analysis and related graphing were carried out using GraphPad Prism (ver.8.0) application. A p value less than 0.05 was considered statistically signifcant.

PANTR1 is Overexpressed in Grade II/III Gliomas.
To elucidate whether PANTR1 is involved in glioma, we compared the expression of PANTR1 in normal adjacent to tumor (NAT) tissues and tumor tissues. Te results showed that expression of PANTR1 was signifcantly increased in several tumor tissues including GBM (Figures 1(a) and 1(b)). Further analysis confrmed a signifcantly higher expression of PANTR1 in glioma tissues (Figures 1(c) and 1(d)). Reverse transcriptionquantitative (RT-q) PCR showed that all the 15 glioma samples' PANTR1 expression outweighs normal adjacent tissues, whereas grade II and III glioma tend to have a higher expression rather than GBM compared with NAT (supplement 10). In the glioma samples of this study, 212 genes (red dots) were found to have closed correlations with PANTR1, while 170 genes (blue dots) were negatively associated. Te top fve genes substantially related to greater PANTR1 expression were EGFR, SEC61G, IGFBP2, and METTL7B. While low expression was related to the downregulation of CAMK2A, SYT1, VSNL1, and PACSIN1 ( Figure 1(e)).

(b)
Te expression of PANTR1 log2 (TPM+1) transporter complex, in biological processes including pattern-specifc process and regionalization, and in molecular functions including substrate-specifc channel activity and gated channel activity (Supplement Tables 1 and 2, Figures 3(a)-3(d)). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment of gene expression in neuroactive ligand-receptor interaction, cAMP signaling pathway, and calcium signaling pathway (Figure 2(e), Supplement Table 3). Based on these results, further KEGG analysis of GBM/LGG samples revealed a signifcant correlation of PANTR1 expression with pathways including mitosis, M-phase, DNA repair, and translation (Supplement Table 3, Figure 4(h)).
Next, immune signatures of GBM/LGG were investigated. Marker genes of 24 immune cells were derived from a study by Bindea et al. [226]. A high expression level of PANTR1 was found to be positively correlated with infltration levels of T2 cells, aDCs, and macrophages. Of note, the expression of PANTR1 was not associated with the infltration of iDCs and NK cells (Figures 4(a)-4(f )).
We also performed an analysis of protein-protein interaction associated with PANTR1, which indicated the involvement of several proteins including EGFR, GRIN2A, HOXA6, and RASGRF1 (Supplement Table 4, Figure 5).

PANTR1 is Associated with Cell Viability, Migration, and
Cell Death. To detect the efects of PANTR1 on gliomas, we used Transwell and transfection assay; it was found that knockdown of PANTR1 was associated with attenuated cell viability and invasiveness (Figures 6(a)-6(d)). Moreover, an increased ratio of G2/M phase and a reduction of S and G0/ G1 phase glioma cells were observed in PANTR1 knockdown group compared with normal glioma cells after 48 and 72 hours (Figures 6(e), 6(f ), and 7(a)-7(d)), which indicated that the SHG44 cell cycle was blocked in G2 phase.
Moreover, according to the result of the wound-healing assay, cells transfected with SiPANTR1 exhibited a perceptible migratory ability decrease after 24 hours compared with control cells (Figure 8(d)). According to the result of the correlation analysis presented by the volcano plot, one of the top fve genes substantially related to the high expression of PANTR1 was EGFR (Figure 1(e)). Te PI3K/ Akt signaling pathway was the major molecular mechanism of EGFR-induced tumorigenic transformation [28]. To further investigate the potential functions of PANTR1 in cell death, we used a western blot assay to detect the PI3K/ Akt signaling pathway. Te result showed that Ser 473 phosphorylated Akt1 expression by the western blot was markedly reduced in PANTR1 knockdown glioma cells, which plays an important role in cell death (Figure 6(g)).
Annexin V/propidium iodide assay also confrmed that in the two cell lines, apoptosis was enhanced after silencing, especially the proportion of late apoptosis and death was increased (Figure 6(h)).
Based on this, a nomogram with variables including WHO grade, IDH status, primary therapy outcome, age, and PANTR1 was constructed (Figure 8(a)). Te receiver operating characteristic (ROC) curve of the nomogram indicated that PANTR1 could serve as a potential diagnostic parameter for gliomas (AUC of 0.958) (Figure 8(b)). Calibration curves for 1-year, 3-year, and 5-year outcomes were depicted in Figure 8(c).

Discussion
In recent years, an accumulating body of research revealed the regulatory role of lncRNAs in tumor treatment. Several lncRNAs were proven to be related to the resistance against glioma treatments, including immunotherapy, chemotherapy, and radiotherapy [11]. Furthermore, Zhang et al. established a lncRNAs signature based on the 16 most potent tumor-infltrating immune cell-associated lncRNAs, which could predict the superior responses of immunotherapy in multiple datasets across cancer types [29]. Wu et al. reported an immune-related lncRNAs signature that could predict the therapeutic efcacy of low-grade glioma treatments [30]. In the current study, we frst found the mRNA level of PANTR1 increased in glioma samples by a public dataset mining and verifed by (RT-q) PCR of collected glioma samples. Te Kaplan-Meier survival analysis showed that PANTR1 was correlated with a poor prognosis. Te in-vitro study showed that the knockdown of PANTR1 caused signifcantly reduced glioma cells viability. In addition, according to the western blot and Annexin V/propidium iodide assay, PANTR1 is highly correlated with the upregulation of the phosphorylation of AKT1 at S473, which promotes cell survival by inhibiting apoptosis via phosphorylation and inactivation of several targets including Bad, Foxo1, c-Raf, and caspase-9. Hence, PANTR1 might be an oncogene in glioma. In addition, wound-healing assay that showed a migratory capacity decline of Si-PANTR1 transfected cells suggested PANTR1 is associated with tumor invasiveness and metastasis. Both genetic and epigenetic mechanisms play important roles in tumorigenesis.
PANTR1 is a nonprotein-coding transcript with four 4KB variants upstream of the POU3F3 gene on chromosome 2q12.1. Several studies have revealed that PANTR1 plays a vital function in the development of the human neurological and urinary systems [20,31,32]. At the epigenetic level, PANTR1 has been proven to be involved in the regulation of the expression of Delta1 and Sox2, both of which are key genes in the neural diferentiation of stem cells [33].

Journal of Oncology
Clinical presentations of POU3F3 mutations vary from cognitive impairment and malformation of several organs [34]. Meanwhile, PANTR1 played an important role in tumorigenesis. Some suggested that the combination of PANTR1 with other lncRNAs may identify the early progression of clear-cell renal cell cancer, breast cancer, and cervical cancer [21,35,36]. It was also demonstrated that the CpG island near the PANTR1 gene is highly methylated and that its transcriptional activity is generally inhibited in the process of tumor progression [37].
Several studies indicated PANTR1 is a driver gene of diferent types of cancers including cervical cancer, clear-cell renal cell cancer, esophageal squamous cell carcinoma, gastric cancer, and breast cancer, as PANTR1 can promote angiogenesis, cell proliferation, migration, and invasion, and inhibit angiogenesis cell-cell death [21,35,36,38,39].
Previous studies demonstrated that Linc-POU3F3 (PANTR1) could promote tumor cell invasion in hepatocellular carcinoma and colorectal cancer [40,41]. In the present study, cell invasion assays showed decreased invasiveness in both SW1088 and SGH44 glioma cell lines after PANTR1 knockdown, which were consistent with the results in other tumor types.
After PANTR1 knockdown, we observed diferent degrees of cell growth and cell death in cell lines, which was more pronounced in low-grade glioma cells. To elucidate the cell behavior pattern observed by cell viability inhibition, we studied the apoptotic activity of PANTR1 knockdown cells.

Journal of Oncology
In the present study, PANTR1 was found to be significantly up-regulated in glioma tissues. Kaplan-Meier survival analysis showed high PANTR1 expressed gliomas indicate poor prognosis. Bioinformatic techniques play a critical role in this study. At the epigenetic level, we demonstrated the upregulation of several genes including EGFR, SEC61G, IGFBP2, and METTL7B by PANTR1.
Pathway analysis by KEGG revealed the regulatory role of PANTR1 in mitosis, M-phase, and translation. In terms of the immune microenvironment, we found that high expression of PANTR1 may be negatively associated with the infltration of iDCs and NK cells. In addition, a major inhibition of cell viability (G2 phase blockage in the cell cycle), migration, and invasiveness was found in PANTR1 knock-out        [21]. Tere are many apoptosis-related pathways, such as PERK and caspase 7 pathways [41,42]. Future experiments could be carried out to validate the role of these pathways in glioma progression. Collectively, the nomogram we constructed proved that PANTR1 could serve as a potential biomarker of glioma prognosis, as high expression of PANTR1 may confer a variety of adverse events at the molecular level, especially in those with grade II/III gliomas. Te role of lncRNAs in tumorigenesis is receiving increasing recognition, our study provides insight into future therapeutic targets for gliomas, and we believe that further studies are still needed to further establish the role of PANTR1.

Data Availability
Publicly available datasets were analyzed in this study. Tis data can be found here: the datasets analyzed for this study can be found on the TCGA GDC ofcial website (https:// portal.gdc.cancer.gov/) and Genomics of Drug Sensitivity in Cancer (GDSC; https://www.cancerrxgene.org/)

Conflicts of Interest
Te authors declare that they have no conficts of fnancial interest.

Authors' Contributions
Fei Shi, Jie Hu, and Ping Zheng contributed to this work equally and shared the frst authorship.

Acknowledgments
Tis work was supported by grants from the Cross Research Fund of Medicine and Engineering of Shanghai Jiao Tong University (grant no. YG2021QN90). Table 1: Diferential expression analysis of PANTR1 in GBM/LGG. Table 2: Gene ontology enrichment analysis of PANTR1 using the clusterProfler package. Table 3: Pathway enrichment analysis of PANTR1. Table 4: Protein-protein interaction network of PANTR1. Table 5: Te association of PANTR1 expression level with clinical parameters of gliomas using the Chi-squared test or Fisher's exact test for analysis. Student's t-test or Wilcoxon rank sum test revealed that age was signifcantly (p < 0.001) associated with PANTR1 expression. Table 6: Te association of PANTR1 expression level with pathological parameters of gliomas using logistics regression. PANTR1 expression was signifcantly correlated with these variables including WHO grade (p < 0.001), IDH status (p < 0.001), primary therapy outcome (p � 0.016), and EGFR status (p < 0.001). Table 7: Uniand multivariate Cox regression analysis showed the prognostic value of PANTR1 in overall survival. We observed IDH status (p < 0.001), primary therapy outcome (p < 0.001), age (p � 0.022), and PANTR1 (p � 0.045) are independent prognostic factors in progression-free interval (p < 0.05) of gliomas. Table 8: Uni-and multivariate Cox regression analysis showed the prognostic value of PANTR1 in progression-free survival.